1. Field of the Invention
The present invention relates to alleles of the lysC gene from corynebacteria that code for desensitized aspartokinases, and processes for the preparation of L-lysine using bacteria containing these alleles.
2. Description of the Background
The amino acid L-lysine is used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.
It is known to prepare amino acids by the fermentation of strains of corynebacteria, especially a Corynebacterium glutamicum. Because of their great importance, attempts are constantly being made to improve the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, for example stirring and oxygen supply, or the composition of the nutrient media, for example the sugar concentration during fermentation, or the work-up to the product form, for example by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites or auxotrophic for metabolites of regulatory significance, and produce amino acids. A known antimetabolite is the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC).
An important reaction step in the biosynthesis of L-lysine is the aspartokinase reaction. The enzyme aspartokinase catalyzes the conversion of L-aspartic acid to aspartylphosphoric acid. The gene coding for aspartokinase is called lysC or ask. The nucleotide sequence of the lysC or ask gene from the wild-type strain Corynebacterium glutamicum ATCC 13032 is known and is described for example in Kalinowski et al. (Molecular Microbiology 5, 1197–204 (1991)) or Follettie (Journal of Bacteriology 175, 4096–4103 (1993)). The activity of the enzyme in the wild-type form is inhibited inter alia by mixtures of lysine and threonine, or mixtures of AEC and threonine, or lysine alone, or AEC alone.
The literature contains mutated forms of the lysC gene which were isolated after mutagenesis with e.g. N-methyl-N′-nitro-N-nitrosoguanidine and selection with the aid of AEC. These lysC alleles code for aspartokinases which, compared with the wild-type form, exhibit a lower sensitivity to inhibition by mixtures of lysine and threonine, or mixtures of AEC and threonine, or lysine alone, or AEC alone. In this connection the aspartokinases are said to be desensitized or feedback-resistant or AEC-resistant. L-lysine-producing strains typically contain such desensitized aspartokinases.
Examples of such desensitized aspartokinases from corynebacteria are described in EP-A-0387527, in U.S. Pat. No. 5,688,671 and in publicly accessible data banks for nucleotide sequences, for example that of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or that of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).
Methods of recombinant DNA technology have also been used for some years to improve L-amino acid-producing strains of Corynebacterium by amplifying individual amino acid biosynthesis genes and studying the effect on amino acid production.